Under the proper conditions, endothelial cells or their precursors/progenitor cells, spontaneously assemble into networks and differentiate to form lumenized connected capillary networks.
Both the forming capillary network and the final capillary network and its associated extracellular matrix (ECM) have numerous scientific, industrial and medical applications. In vitro angiogenesis assays employing the forming capillary network (or capillary fabrication devices) play a crucial role in identifying factors involved in vascular development. Such assays are used in drug development as moderate-throughput screens for angiogenesis promoters and inhibitors related to wound healing, age-related macular degeneration, diabetes, cancer, and other diseases.
The completed networks formed by in vitro angiogenesis devices are also important in tissue engineering, as vascular replacements for human implantation, while the ECM of the networks can be employed as a scaffold implanted into patients to promote the growth of the patient's own vasculature into the pattern provided by the ECM.
Drug development and tissue engineering applications both require rapid creation of viable lumenized capillary networks having properties as close as possible to in vivo capillary networks. Existing in vitro angiogenesis techniques are not capable of rapidly creating lumenized capillary networks with properties substantially similar to in vivo networks.
Current methods of tissue engineering are limited to relatively thin and/or avascular tissues like skin, cartilage, and bladder, where post-implantation vascularization from the host is sufficient to meet the implant's demand for oxygen and nutrients. Vascularization remains a critical obstacle for engineering thicker, metabolically demanding organs, such as heart and liver. Thus the survival of tissue-engineered three-dimensional constructs in vivo depends on providing enough blood supply to the engineered tissue and the capacity of the engineered microcirculation to connect with the existing circulation of the recipient.
Engineering thicker, metabolically demanding organs, such as heart and liver, requires techniques for manufacturing microvessels with highly controlled geometries. Existing microcirculation engineering techniques are not capable of constructing controlled-geometry microvessels capable of connection to the host vessels, expansion of vascular volume accompanying growing tissue, and prevention of excessive vascular regression.
Current in vitro capillary fabrication devices are either quasi-two-dimensional or three-dimensional. Quasi-two-dimensional devices are categorized as either rapid or long-term. Rapid quasi-two-dimensional devices consist of a layer of endothelial cells seeded sub-confluently on top of a thick layer of a basement-membrane gel which is made of a mixture of collagen, fibrin, or Matrigel™. Depending on the components and mechanical properties of the gel, endothelial cells align to form a one-cell thick capillary-like pattern within 1 to 3 days. In rapid devices, the standard Matrigel™ capillary fabrication device (FIG. 4) is widely used, especially in assaying applications to characterize anti-angiogenic or pro-angiogenic factors. In the standard quasi-two-dimensional Matrigel™ capillary fabrication device, cells plated on a thick layer (about 0.5 mm) of Matrigel™ are exposed to high levels of soluble and ECM-bound growth factors. A high concentration of growth factors in Matrigel™ (or even Growth Factor Reduced (GFR) Matrigel™) results in artifacts and over-stimulation of cells. Further, cell motility is restricted by adhesion of cells to the solid Matrigel™ and resultant capillaries have endothelial cells that are abnormally elongated compared to the in vivo morphology of endothelial cells. The endothelial cells typically die 24 to 48 hours after forming the networks in rapid quasi-two-dimensional devices (Ranta et al. (1998), J Cell Physiol. 176(1):92-98; Vailhé et al. (2001) Lab Invest. 81(4):439-452); thus these devices are not suitable for applications requiring viable capillary networks. However, the lumenized capillary networks formed in the devices disclosed herein remain viable for about 4 weeks.
Long-term quasi-two-dimensional devices generally consist of endothelial cells suspended in normal culture medium and conditions without inclusion of an extracellular matrix substrate. As the endothelial cells divide, they form a confluent mono-layer, after which some differentiate spontaneously to form capillary-like structures on top of a confluent layer of undifferentiated endothelial cells. Long-term devices require 2 to 8 weeks of cell culture, making them unsuitable for high-throughput experiments.
Applications of quasi-two-dimensional angiogenesis devices include, for example, studies of the role and synthesis of extracellular matrix in vascular morphogenesis; studies of the roles of adhesion molecules; screening of angiostatic molecules; functional characterization of endothelial cell lines; studies of proteases; extracellular protein synthesis; vessel maturation; and studies of the role of glycation products in diabetes.
Three-dimensional capillary fabrication devices combine endothelial cells with a three-dimensional gel which the endothelial cells then invade. Widely-used three-dimensional in vitro devices include aortic rings in gelified matrices, endothelial cells seeded inside a gel or sandwiched between two layers of gel or between a single layer of gel and a cell-culture surface, and microcarrier beads coated with endothelial cells. The forming capillaries in three-dimensional angiogenesis devices are applied to study the effects of cytokines, metalloproteases and the fibrinolytic pathway during tubulogenesis, endothelial apoptosis, the importance of the configuration and composition of the substrate, the role of cell-adhesion molecules, the effect of hypoxia on endothelial cells, and for screening of angiogenic and angiostatic molecules.
Three-dimensional capillary fabrication devices require many more cells to achieve the cell densities required for fabrication of connected capillary networks. Cell distribution in three-dimensional capillary fabrication devices is not uniform which may cause over-crowded regions and increased cell death. The transport of oxygen and other nutrients to three-dimensional matrices via diffusion limits the thickness of the three-dimensional matrices in those capillary fabrication devices. The cell resources in capillary fabrication devices are prohibitively limited and expensive. Since the cells are embedded in a solidified gel which limits cell motility, the formation of capillary-like patterns is slow, taking 1 to 8 weeks. Thus three-dimensional capillary fabrication devices are inefficient and slow which make them not suitable for industrial, drug-development and high throughput applications.
Thus, improvements are needed for in vitro capillary fabrication devices.
Techniques for the vascularization of tissue-engineered constructs can be broadly grouped into in vitro and in vivo approaches. In vivo approaches rely on vessel ingrowth from host to the engineered tissue. This ingrowth process is often limited to tenths of microns per days, meaning that the time needed for complete vascularization of an implant of several millimeters is in the order of weeks. Several strategies have been developed for improving the ingrowth of vessels from host tissue including scaffold designs, angiogenic-factor delivery and in vivo pre-vascularization. In vitro approaches include in vitro pre-vascularization techniques. In vitro approaches do not rely on ingrowth of host vessels into entire construct. However, anastomosis of the vessels in the in vitro-pre-vascularized tissues is not as fast as in vivo pre-vascularization. In vitro-pre-vascularization also needs to have proper organization and geometry to be able to provide enough blood perfusion after implantation of the engineered tissue.
The capillary fabrication devices disclosed herein can produce highly controlled functional lumenized capillary networks that enhance blood perfusion in the engineered tissue.
Disclosed herein are capillary fabrication devices for manufacturing forming capillary networks and formed capillary networks with natural or controlled geometries the engineered ECM and basement membrane associated with the capillary networks, and application of the capillary networks and ECM and basement membrane for construction of engineered tissues.
The capillary fabrication devices described herein produce capillaries which improve in a number of ways on the standard quasi-two-dimensional Matrigel™ capillary fabrication device. Unlike the standard quasi-two-dimensional Matrigel™ capillary fabrication device, which produces a capillary-like structure composed of unnaturally elongated endothelial cells, the morphology of endothelial cells incorporated in the capillary cords and resulting lumens in the capillary fabrication device described herein are very similar to those in capillaries formed in vivo, e.g., in zebrafish embryos and chick allantois.
It has also been found that the standard quasi-two-dimensional Matrigel™ capillary fabrication device (even when growth-factor reduced Matrigel™ is used) produces a honeycomb-like network, in which tubules are often composed of a single endothelial cell stretched and connected to aggregates of endothelial cells. Most capillary tubules formed in the capillary fabrication devices described herein are composed of several cell lengths, and are thus capable of producing mean tubule lengths comparable to capillaries in vivo (FIG. 1). Capillary networks produced using the standard quasi-two-dimensional Matrigel™ capillary fabrication device are not viable for more than 48 hours. The lumenized capillary networks of the capillary fabrication devices described herein may be viable for up to 4 weeks after formation of the network.
The standard quasi-two-dimensional Matrigel™ capillary fabrication device is unable to recapitulate normal cell motility and proliferation since endothelial cells plated on top of a thick layer of Matrigel™ show no or little proliferation and motility. The cells do not have properties that are substantially similar to in vivo cells. For example, mean tubule diameter which has significant biological importance, is usually less than 5 microns in the standard quasi-two-dimensional Matrigel™ capillary fabrication device. Formation of narrow tubes (inner diameter<4 microns) in the standard quasi-two-dimensional Matrigel™ capillary fabrication device also indicates high levels of mechanical stress in the individual cells. Tubes less than 4 microns in diameter do not support natural blood flow. Thus they are non-functional. However, the diameter of engineered lumenized capillaries produced using the capillary fabrication devices described herein were found to range from 5 to 20 microns, which matches the capillary diameters of zebrafish embryos, chick allantois and many human tissues.
In the capillary fabrication devices described herein, only endothelial lineage cells incorporated in capillary cords are not motile and show reduced proliferation potency because of strong junctional complexes; the rest of the cells proliferate and are motile. Thus, the capillary fabrication devices described herein are less sensitive to the seeded cell density than the standard quasi-two-dimensional Matrigel™ capillary fabrication device. This allows formation of capillary networks from limited numbers of stem cells or from an autograft. An autograft capillary network has a lower chance of rejection in tissue repair and engineering applications.
In the capillary fabrication devices described herein, similar to long-term in vitro devices, the lumenized capillary networks remain viable for at least one week after tubulogenesis, and often remain viable for four weeks or more. In contrast, in most in vitro capillary fabrication devices, the capillary-like networks degrade and disappear after tubulogenesis. The longer viability of the engineered capillary networks described herein allows for assays to study the effects of pro/anti-angiogenic factors on both established lumenized capillary networks and the initial stages of tubulogenesis. The steps of endothelial-cell tubulogenesis can be recapitulated in quasi-in vivo conditions.
Use of cell-culture-treated dishes, rather than non-treated dishes, does not produce a capillary-like pattern, but rather increases proliferation of seeded endothelial resulting in a confluent layer of undifferentiated endothelial cells. Typically, normally adherent cells cultured in non-treated polystyrene dishes without coating undergo anoikis/apoptosis after a few hours and die. However, in the capillary fabrication device disclosed herein, the use of a support-generating medium to form the support medium on a non-treated polystyrene surface as a cell-culture surface allows the cells to survive and form capillary networks. Without being bound by theory, it is believed that the binding of specific integrin receptors in the endothelial cells contribute to the initiation of transcription of anti-apoptotic genes, differentiation and tubulogenesis.
Current methods of tissue engineering are limited by the difficulties of forming functional vascular networks in the engineered tissues. Engineered blood vessels using prosthetic conduits are not suitable for vessel diameters of less than 6 mm due to formation of thromboses. Engineered blood vessels which have a lining of endothelial cells, using cell-sheet engineering or bioprinting, are limited to large blood vessels. Thus existing tissue engineering methods are unable to produce functional vascular networks for tissue engineering. The capillary fabrication device and custom-patterned capillary fabrication device are capable of producing functional capillaries which can be integrated with larger vessels in the host tissue or engineered tissue to form functional vascular networks. Engineering thicker metabolically demanding organs, such as heart and liver requires techniques for manufacturing microvessels with highly controlled geometries. Existing microcirculation engineering techniques are not capable of constructing controlled-geometry microvessels capable of connecting to the host vessels, expansion of vascular volume accompanying growing tissue, and prevention of excessive vascular regression. The custom-patterned/controlled-geometry capillary device is capable of producing functional lumenized capillary networks which can highly optimize blood perfusion in the engineered tissue.
In medically-oriented tissue engineering applications autologous resources of cells are limited. Thus engineering tissues from autologous components is often not practical. Extracellular components are highly conserved and known to enhance and regulate growth/regrowth and differentiation of cells in engineered tissues. Use of organically-fabricated ECM and basement membrane (formed using the devices disclosed herein) of non-autologous or non-human (like mouse) origin can be useful for medical tissue-repair and tissue engineering applications with minimal transfer of external factors and thus significant reduction of the likelihood of rejection after implant.
The following various embodiments are provided:
1) A device for fabrication of engineered capillary networks comprising:
living cells;
a cell-culture surface; and
a support-generating medium, the support-generating medium comprising a gel forming material and a liquid cell-culture medium, wherein the gel forming material is substantially dissolved in the cell-culture medium and forms a support medium on the cell-culture surface.
2) A custom-patterned capillary fabrication device comprising:
living cells;
a cell-culture surface; and a support-generating medium, the support-generating medium comprising a gel forming material and a liquid cell-culture medium, wherein the gel forming material is substantially dissolved in the cell-culture medium and forms a support medium on the cell-culture surface, and wherein the cell-culture surface comprises a network-like pattern containing regions of varying hydrophobicity.
3) The device of any of clauses 1 to 2 wherein the cells are of human origin.
4) The device of any of clauses 1 to 3 wherein the living cells are of endothelial lineage.
5) The device of any of clauses 1 to 4 wherein the cells are selected from the group consisting of embryonic stem cells, endothelial progenitor cells, circulating endothelial cells, and lymphatic endothelial cells.
6) The device of any of clauses 4 to 5 further comprising at least one additional cell type.
7) The device of clause 6 wherein the additional cell type is selected from the group consisting of pericytes, smooth muscle cells, fibroblasts, and any combination thereof.
8) The device of any of clauses 1 to 7 wherein one or more cells are modified cells.
9) The device of any of clauses 1 to 8, wherein the cell-culture surface comprises at least one hydrophobic region.
10) The device of any of clauses 1 to 9 wherein the cell-culture surface comprises a coating of at least one temperature sensitive polymer.
11) The device of clause 10 wherein at least one temperature sensitive polymer is poly(N-isopropylacrylamide).
12) The device of any of clauses 1 to 11 wherein the cell-culture surface is flat.
13) The device of clause 12 wherein the cell-culture surface is selected from the group consisting of petri dishes, well-plates, slides, and coverslips.
14) The device of any of clauses 1 to 13 wherein the cell-culture surface is comprised of a material selected from the group consisting of non-treated polystyrene, glass, a temperature sensitive polymer, and any combination thereof.
15) The device of any of clauses 1 to 14 wherein the cell-culture surface further includes meshes or scaffolds.
16) The device of any of clauses 1 to 15 wherein the cell-culture surface is modified by etching, stamping, contact printing, UV laser ablation, or any combination thereof.
17) The device of any of clauses 1 to 16 wherein the cell-culture medium is a defined cell-culture medium.
18) The device of any of clauses 1 to 16 wherein the cell-culture medium comprises serum albumin.
19) The device of any of clauses 1 to 18 wherein the cell-culture medium comprises a bicarbonate-base or HEPES buffer.
20) The device of any of clauses 1 to 19 wherein the gel-forming material comprises at least one extracellular matrix (ECM) protein.
21) The device of any of clauses 1 to 20 wherein the gel forming material comprises at least one protein selected from the group consisting of laminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen, TGF-β, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, and tissue plasminogen activator.
22) The device of any of clauses 1 to 20 wherein the gel-forming material is Matrigel™.
23) The device of any of clauses 1 to 20 wherein the support-generating medium comprises Matrigel™ diluted from 1 to 30 to about 1 to 60 times in the liquid cell-culture medium.
24) The device of any of clauses 1 to 20 wherein the support-generating medium is dissolved in the liquid-cell culture medium to yield an ECM protein concentration of from about 170 μg ECM proteins per ml of liquid-cell culture medium to about 350 μg ECM proteins per ml of liquid-cell culture medium.
25) The device of any of clauses 1 to 24 wherein the support medium harbors the living cells.
26) The device of any of clauses 1 to 25 wherein the support medium has a thickness of less than 150 microns.
27) The device of any of clauses 1 to 25 wherein the support medium has a thickness of less than 50 microns.
28) The device of any of clauses 1 to 25 wherein the support medium has a thickness from 50 microns to 100 microns.
29) The device of any of clauses 1 to 25 wherein the support medium has a thickness from 20 microns to 40 microns.
30) A formed capillary network made using the device according to any of clauses 1 to 29.
31) The capillary network of clause 30 wherein the capillary network comprises tubules having a diameter of at least 5 microns.
32) The capillary network of clause 30 wherein the capillary network comprises tubules having a diameter of at least 10 microns.
33) The capillary network of clause 30 wherein the capillary network comprises tubules having a diameter of at least 15 microns.
34) The capillary network of clause 30 wherein the capillary network comprises tubules having a diameter of at least 20 microns.
35) The capillary network of any of clauses 30 to 34 wherein the capillary network geometry is pre-patterned.
36) Use of the device according to any of clauses 1 to 29 to generate a formed capillary network.
37) Use of the device of any of clauses 1 to 29 to generate a forming capillary network.
38) The use according to any of clauses 36 to 37 wherein the living cells are contacted by a test substance suspected to promote or inhibit angiogenesis.
39) An ECM and basement membrane produced by the living cells of the device according to any of clauses 1 to 29, wherein the ECM and basement membrane are detached from a forming or formed capillary network.
40) Use of the device according to any of clauses 1 to 29 to generate an ECM and basement membrane that are detachable from a forming or formed capillary network.
41) The use according to clause 40 wherein the ECM and basement membrane are detached from the capillary network by at least changing the cell-culture temperature.
42) The use according to any of clauses 40 to 41 wherein the ECM and basement membrane are detached from the capillary network by at least changing the pH of the cell-culture medium.
43) The use according to any of clauses 40 to 42 wherein the ECM and basement membrane are detached from the capillary network by at least contacting the capillary network and ECM and basement membrane with a stream of liquid.
44) Use of a capillary network produced by the device of any of clauses 1 to 29 to repair or regenerate tissue in vivo.
45) Use of the ECM and basement membrane of clause 39 in an in vitro tissue engineering application.
46) Use of the ECM and basement membrane of clause 39 in a cell-sheet engineering application.
47) Use of the ECM and basement membrane in a bioprinting application.